Non-small cell lung cancer (NSCLC) is the most common cause of premature death from malignant disease in western countries. Early detection followed by surgery remains the most effective treatment. The diagnosis of patients who have lung nodules of unknown etiology identified on screening chest X-rays or computerized axial tomography (CT) scans, is a very common and important clinical problem for internists, surgeons, and pulmonologists. A blood test that could more accurately determine the risk of malignant disease in patients with lung nodules would be extremely valuable and have very important economic implications by reducing unnecessary surgery, biopsies, Positron Emission Tomography (PET) scans, and/or repeated CT scans. We previously hypothesized that a diagnostic gene expression profile can be identified in the peripheral blood of lung cancer patients. In support of this hypothesis, we recently published a study showing that differences in blood gene expression profiles can be identified that accurately identify patients with NSCLC when compared to the appropriate control group of high-risk smokers and ex-smokers. Using peripheral blood mononuclear cells (PBMC) harvested from 137 NSCLC subjects and 91 controls, we identified a 29-gene signature that provides an overall discrimination accuracy of 86%. Specific immune function pathways that were affected in the NSCLC group included alterations in T-cell receptor signaling and apoptosis, and macrophage and monocyte phagocytosis. The goal of this proposal is to expand these preliminary studies to the Paxgene blood collection system, which provides greater mRNA stability and a platform more easily adopted for routine clinical use. We will focus our work on developing a peripheral blood gene expression signature from peripheral blood with the Paxgene system (Aim 1) and on evaluating the combination of peripheral blood gene expression with multiple novel serum biomarkers (Aim 2). If successful, these assays could be integrated into a diagnostic algorithm for patients with indeterminate lung nodules and used to prevent needless surgery and repetitive CT scans. If sufficiently sensitive and specific, it is possible that this test could be extended to screening of other high-risk populations (smokers with asbestos exposure, heavy smokers, etc).